Phosphorylation and activation of p40 tyrosine kinase by casein kinase-1

Because examination of regulatory trans-phosphorylations can help elucidate the cellular functions of tyrosyl protein kinases, we have investigated the effects of phosphorylation by casein kinase-1 on the activity of the p40 tyrosyl protein kinase. We find that casein kinase-1 can phosphorylate the p40 tyrosyl kinase on serine and threonine residues, in part on a unique tryptic peptide. The phosphorylation induces a substantial increase in the tyrosyl protein kinase activity of p40, in contrast to most instances in which serine/threonine phosphorylation inhibits activity of tyrosyl protein kinases. These findings raise the possibility that p40 might be part of a protein phosphorylation network in which casein kinase-1 participates.     

Evidence that a eukaryotic-type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division.

A eukaryotic-type protein serine/threonine kinase, PknA, was cloned from Mycobacterium tuberculosis strain H37Ra. Sequencing of the clone indicated 100% identity with the published pknA sequence of M. tuberculosis strain H37Rv. PknA fused to maltose-binding protein was expressed in Escherichia coli; it exhibited a molecular mass of approximately 97 kDa. The fusion protein was purified from the soluble fraction by affinity chromatography using amylose resin. In vitro kinase assays showed that the autophosphorylating ability of PknA is strictly magnesium/manganese-dependent, and sodium orthovanadate can inhibit this activity. Phosphoamino-acid analysis indicated that PknA phosphorylates at serine and threonine residues. PknA was also able to phosphorylate exogenous substrates, such as myelin basic protein and histone. A comparison of the nucleotide-derived amino-acid sequence of PknA with that of functionally characterized prokaryotic serine/threonine kinases indicated its possible involvement in cell division/differentiation. Protein--protein interaction studies revealed that PknA is capable of phosphorylating at least a approximately 56-kDa soluble protein from E. coli. Scanning electron microscopy showed that constitutive expression of this kinase resulted in elongation of E. coli cells, supporting its regulatory role in cell division.     

Recombinant human pim-1 protein exhibits serine/threonine kinase activity.

The protein predicted by the sequence of the human pim-1 proto-oncogene shares extensive homology with known serine/threonine protein kinases, and yet the human Pim-1 enzyme has previously been reported to exhibit protein tyrosine kinase activity both in vitro and in vivo. Recently a new class of protein kinases has been identified which exhibits both protein-serine/threonine and protein-tyrosine kinase activities. We therefore investigated the possibility that the human Pim-1 kinase likewise possesses such bifunctional enzymatic phosphorylating activities. A full-length human pim-1 cDNA was subcloned into the bacterial vector pGEX-2T and the Pim-1 protein expressed as a fusion product with bacterial glutathione S-transferase (GST). The hybrid GST-Pim-1 fusion protein was affinity purified on a glutathione-Sepharose column prior to treatment with thrombin for cleavage of the Pim-1 protein from the transferase. Pim-1 was purified and the identity of recombinant protein confirmed by amino-terminal sequence analysis. Pim-1 was tested for kinase activity with a variety of proteins and peptides known to be substrates for either mammalian protein-serine/threonine or protein-tyrosine kinases and was found to phosphorylate serine/threonine residues exclusively in vitro. Both the Pim-1-GST fusion protein and the isolated Pim-1 protein exhibited only serine/threonine phosphorylating activity under all in vitro conditions tested. Pim-1 phosphorylated purified mammalian histone H1 with a Km of approximately 51 microM. Additionally, Pim-1 exhibited low levels of serine/threonine autophosphorylating activity. These observations place the human Pim-1 in a small select group of cytoplasmic transforming oncogenic kinases, including the protein kinase C, the Raf/Mil, and the Mos subfamilies, exhibiting serine/threonine phosphorylating activity.     

Phosphorylation of Microtubule-Associated Protein 2 at Distinct Sites by Calmodulin-Dependent and Cyclic-AMP-Dependent Kinases

Microtubule-associated protein 2 (MAP2) is an excellent substrate for both cyclic-AMP (cAMP)-dependent and Ca2+/calmodulin-dependent kinases. A recently purified cytosolic Ca2+/calmodulin-dependent kinase (now designated CaM kinase II) phosphorylates MAP2 as a major substrate. We now report that microtubule-associated cAMP-dependent and calmodulin-dependent protein kinases phosphorylate MAP2 on separate sites. Tryptic phosphopeptide digestion and two-dimensional phosphopeptide mapping revealed 11 major peptides phosphorylated by microtubule-associated cAMP-dependent kinase and five major peptide species phosphorylated by calmodulin-dependent kinase. All 11 of the cAMP-dependently phosphorylated peptides were phosphorylated on serine residues, whereas four of five major peptides phosphorylated by the calmodulin-dependent kinase were phosphorylated on threonine. Only one peptide spot phosphorylated by both kinases was indistinguishable by both migration and phosphoamino acid site. The results indicate that cAMP-dependent and calmodulin-dependent kinases may regulate microtubule and cytoskeletal dynamics by phosphorylation of MAP2 at distinct sites.     

WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II

We have cloned and characterized a novel mammalian serine/threonine protein kinase WNK1 (with no lysine (K)) from a rat brain cDNA library. WNK1 has 2126 amino acids and can be detected as a protein of approximately 230 kDa in various cell lines and rat tissues. WNK1 contains a small N-terminal domain followed by the kinase domain and a long C-terminal tail. The WNK1 kinase domain has the greatest similarity to the MEKK protein kinase family. However, overexpression of WNK1 in HEK293 cells exerts no detectable effect on the activity of known, co-transfected mitogen-activated protein kinases, suggesting that it belongs to a distinct pathway. WNK1 phosphorylates the exogenous substrate myelin basic protein as well as itself mostly on serine residues, confirming that it is a serine/threonine protein kinase. The demonstration of activity was striking because WNK1, and its homologs in other organisms lack the invariant catalytic lysine in subdomain II of protein kinases that is crucial for binding to ATP. A model of WNK1 using the structure of cAMP-dependent protein kinase suggests that lysine 233 in kinase subdomain I may provide this function. Mutation of this lysine residue to methionine eliminates WNK1 activity, consistent with the conclusion that it is required for catalysis. This distinct organization of catalytic residues indicates that WNK1 belongs to a novel family of serine/threonine protein kinases.     

Analysis of the protein kinase activity of moss phytochrome expressed in fibroblast cell culture

In the moss Ceratodon purpureus a phytochrome gene encodes a phytochrome type (PhyCer) which has a C-terminal domain homologous to the catalytic domain of eukaryotic protein kinases (PKs). PhyCer exhibits sequence conservation to serine/ threonine as well to tyrosine kinases. Since PhyCer is expressed very weakly in moss cells, to investigate the proposed PK activity of PhyCer, we overexpressed PhyCer transiently in fibroblast cells. For this purpose we made a chimeric receptor, EC-R, which consists of the extracellular, the membrane-spanning and the juxtamembrane domains of the human epidermal growth-factor receptor (EGF-R) linked to the PK catalytic domain of PhyCer (CerKin). The expression of EC-R in transiently transfected cells was confirmed with antibodies directed against the extracellular domain of EGF-R or against CerKin. Both EGF-R and EC-R were immunoprecipitated from lysates of overexpressing cells with antibodies against the extracellular domain of EGF-R. Phosphorylation experiments were performed with the immunoprecipitates and the phosphorylation products were subjected to phosphoamino acid analysis. Phosphorylation products specifically obtained with EC-R-transfected cells exhibit phosphorylation on serine and threonine residues. In EC-R transfected cells the endogenous EGF-R showed enhanced phosphorylation of serine and threonine residues compared to EGF-R immuno-precipitated from control cells. Although CerKin is closest to the catalytic domain of a protein tyrosine kinase from Dictyostelium discoideum, EC-R does not appear to phosphorylate tyrosine residues in vitro. From our data we conclude that PhyCer carries an active PK domain capable of phosphorylating serine and threonine residues.Abbreviations CerKin protein kinase catalytic domain of PhyCer - EC-R chimeric receptor consisting of the extracellular, the membrane spanning and the juxtamembrane domains of the human epidermal growth factor receptor (EGF-R) linked to the protein kinase catalytic domain of PhyCer - EGF-R epidermal growth factor receptor - mAb monoclonal antibody - PhyCer phytochrome gene in Ceratodon encoding a phytochrome type which has a C-terminal domain homologous to the catalytic domain of eucaryotic protein kinases - PK protein kinase - PVDF polyvinyl difluoride - Ser serine - Thr threonine - Tyr tyrosine Dr. Patricia Algarra was supported by the Alexander von Humboldt Foundation, Germany. This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany.     

Angiotensin II stimulates the pp44 and pp42 mitogen-activated protein kinases in cultured rat aortic smooth muscle cells.

Vasoconstrictors such as angiotensin II (ang II) stimulate vascular smooth muscle cell growth and share many signal transduction mechanisms with growth factors. Recently, growth factors have been shown to stimulate mitogen-activated protein (MAP) kinases, a family of serine/threonine protein kinases which phosphorylate pp90rsk, a cytosolic kinase that phosphorylates ribosomal S6 protein. We examined the effect of ang II on MAP kinase activity and phosphorylation. Ang II stimulated MAP kinase activity by 4-fold after 5 min exposure and also increased tyrosine phosphorylation of 42 kDa (74 +/- 41%) and 44 kDa (263 +/- 85%) proteins, shown to be pp42mapk and pp44mapk by Western blot analysis using a MAP kinase antibody. These results suggest that ang II-stimulated protein synthesis is mediated by a MAP kinase dependent pathway.     

Identification of a novel casein kinase activity in HeLa cell nuclei

Three casein kinase activities have been resolved by column chromatography of HeLa cell nuclear extracts. In addition to casein kinases NI and NII, which have been described in other cell types, HeLa nuclei contain a third casein kinase activity which we have named NIII. NIII is a cyclic nucleotide-independent casein kinase which uses either Mg2+ or Mn2+ as a divalent cation, but is inhibited by increasing NaCl concentrations in the presence of Mg2+ and has optimal activity at 50 mM NaCl in the presence of Mn2+. In Mg2+, NIII uses only ATP as a phosphate donor, but in Mn2+ NIII transfers phosphate from either ATP or GTP. NIII phosphorylates the serine and threonine residues of casein, but does not phosphorylate phosvitin or calf thymus histones.     

Phosphorylation of microtubule-associated protein 2 by MAP kinase primarily involves the projection domain

Chymotryptic digestion was used to localize the sites in microtubule-associated protein 2 which are preferentially phosphorylated in vitro by MAP kinase, an insulin-stimulated serine/threonine kinase which efficiently utilizes high molecular weight MAPs as substrates. MAP kinase phosphorylates sites in the projection domain almost exclusively; less than 6% of the phosphate incorporated by MAP kinase was found in the tubulin binding domain. This site specificity is in marked contrast to that of the catalytic subunit of cAMP dependent protein kinase, and most other protein kinases phosphorylating MAP-2, which extensively phosphorylate the tubulin binding domain.     

Mutational analysis of the phosphorylation sites of the Aie1 (Aurora-C) kinase in vitro

We previously reported two novel serine/threonine kinases, Aie1 (mouse) and AIE2 (human), both later referred to as aurora-C, a newly recognized member of the mitotic aurora kinase family. In the present study, we analyzed the phosphorylation sites of mouse Aie1 by site-directed mutagenesis. Our results showed that protein kinase A (PKA) phosphorylates Aie1 at a threonine residue located at amino acid position 171. The T171A and T175A mutants, in which threonines located at residues 171 and 175 were replaced by alanines, revealed a significant increase in their kinase activities to phosphorylate ACS-1 (Aurora-C substrate 1). In contrast, the double mutant T171A-T175A showed impaired kinase activity. In addition, we had previously identified a PEST-like motif located at the N terminus of Aie1. Mutation analysis in the present study revealed that the quadruple mutant in which the PEST-like motif was mutated significantly abrogated Aie1 kinase activity. This is the first report of the analysis of potential phosphorylation sites of mouse aurora-C in vitro.     

Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine.

A Saccharomyces cerevisiae lambda gt11 library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase. A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases. We tested the activity of the putative kinase by constructing a vector encoding a glutathione-S-transferase fusion protein containing most of the predicted polypeptide. The fusion protein phosphorylated endogenous substrates and enolase primarily on serine and threonine. The gene was designated SPK1 for serine-protein kinase. Expression of the Spk1 fusion protein in bacteria stimulated serine, threonine, and tyrosine phosphorylation of bacterial proteins. These results, combined with the antiphosphotyrosine immunoreactivity induced by the kinase, indicate that Spk1 is capable of phosphorylating tyrosine as well as phosphorylating serine and threonine. In in vitro assays, the fusion protein kinase phosphorylated the synthetic substrate poly(Glu/Tyr) on tyrosine, but the activity was weak compared with serine and threonine phosphorylation of other substrates. To determine if other serine/threonine kinases would phosphorylate poly(Glu/Tyr), we tested calcium/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The two kinases had similar tyrosine-phosphorylating activities. These results establish that the functional difference between serine/threonine- and tyrosine-protein kinases is not absolute and suggest that there may be physiological circumstances in which tyrosine phosphorylation is mediated by serine/threonine kinases.     

Interaction of the insulin receptor kinase with serine/threonine kinases in vitro

Insulin causes rapid phosphorylation of the beta subunit (Mr = 95,000) of its receptor in broken cell preparations. This occurs on tyrosine residues and is due to activation of a protein kinase which is contained in the receptor itself. In the intact cell, insulin also stimulates the phosphorylation of the receptor and other cellular proteins on serine and threonine residues. In an attempt to find a protein that might link the receptor tyrosine kinase to these serine/threonine phosphorylation reactions, we have studied the interaction of a partially purified preparation of insulin receptor with purified preparations of serine/threonine kinases known to phosphorylate glycogen synthase. No insulin-dependent phosphorylation was observed when casein kinases I and II, phosphorylase kinase, or glycogen synthase kinase 3 was incubated in vitro with the insulin receptor. These kinases also failed to phosphorylate the receptor. By contrast, the insulin receptor kinase catalyzed the phosphorylation of the calmodulin-dependent kinase and addition of insulin in vitro resulted in a 40% increase in this phosphorylation. In the presence of calmodulin-dependent kinase and the insulin receptor kinase, insulin also stimulated the phosphorylation of calmodulin. Phosphoamino acid analysis showed an increase of phosphotyrosine content in both calmodulin and calmodulin-dependent protein kinase. These data suggest that the insulin receptor kinase may interact directly and specifically with the calmodulin-dependent kinase and calmodulin. Further studies will be required to determine if these phosphorylations modify the action of these regulatory proteins.     

The 93 kDa protein gephyrin and tubulin associated with the inhibitory glycine receptor are phosphorylated by an endogenous protein kinase.

The 93 kDa protein gephyrin is a tubulin binding peripheral membrane protein that is associated with the inhibitory glycine receptor and has been implicated in its anchoring at central synapses. Here, we demonstrate that gephyrin as well as co-purifying tubulin are phosphorylated by a kinase activity which is endogenous to highly purified glycine receptor preparations. This kinase phosphorylates serine and threonine residues and utilizes ATP, but not GTP, as phosphate donor. Its activity is not affected by various activators and/or inhibitors of cyclic nucleotide-dependent kinases, calcium/calmodulin-dependent kinases, or protein kinase C. A five-fold stimulation of kinase activity was, however, observed in the presence of poly-lysine. Phosphorylation of gephyrin and/or tubulin might regulate receptor/cytoskeleton interactions at postsynaptic membrane specializations.     

Substrate binding to Src: A new perspective on tyrosine kinase substrate recognition from NMR and molecular dynamics

Most signal transduction pathways in humans are regulated by protein kinases through phosphorylation of their protein substrates. Typical eukaryotic protein kinases are of two major types: those that phosphorylate‐specific sequences containing tyrosine (~90 kinases) and those that phosphorylate either serine or threonine (~395 kinases). The highly conserved catalytic domain of protein kinases comprises a smaller N lobe and a larger C lobe separated by a cleft region lined by the activation loop. Prior studies find that protein tyrosine kinases recognize peptide substrates by binding the polypeptide chain along the C‐lobe on one side of the activation loop, while serine/threonine kinases bind their substrates in the cleft and on the side of the activation loop opposite to that of the tyrosine kinases. Substrate binding structural studies have been limited to four families of the tyrosine kinase group, and did not include Src tyrosine kinases. We examined peptide‐substrate binding to Src using paramagnetic‐relaxation‐enhancement NMR combined with molecular dynamics simulations. The results suggest Src tyrosine kinase can bind substrate positioning residues C‐terminal to the phosphoacceptor residue in an orientation similar to serine/threonine kinases, and unlike other tyrosine kinases. Mutagenesis corroborates this new perspective on tyrosine kinase substrate recognition. Rather than an evolutionary split between tyrosine and serine/threonine kinases, a change in substrate recognition may have occurred within the TK group of the human kinome. Protein tyrosine kinases have long been therapeutic targets, but many marketed drugs have deleterious off‐target effects. More accurate knowledge of substrate interactions of tyrosine kinases has the potential for improving drug selectivity.     

Okadaic acid stimulates the activity of the nerve growth factor-sensitive S6 kinase of PC12 cells.

PC12 pheochromocytoma cells contain at least two different and separable kinases that phosphorylate the S6 protein of the ribosomes. The activity of one of these S6 kinases is increased by treatment of the cells with nerve growth factor and of the other by treatment with epidermal growth factor. Okadaic acid increases the activity of the nerve growth factor-sensitive S6 kinase. The data suggest that the nerve growth factor-sensitive S6 kinase is activated by phosphorylation on serine or threonine residues and is inactivated by either phosphatase 1 or phosphatase 2A, probably the latter.